The present invention relates to a bacterial fermentation process for producing a recombinant protein wherein certain feed media nutrients are monitored and adjusted so as to control metallophosphate precipitation in the media. In particular, the invention is to a high cell density fermentation process comprising a method wherein certain polyphosphates and/or metaphosphates are used in the media to eliminate nutrient precipitation and increase cell density.
Advances in molecular biology and the exploitation of recombinant DNA procedures has made possible the production of significant quantities of foreign proteins in certain host cell systems. Recombinant proteins are produced in the host cell systems by transfecting the host cells with DNA coding for the protein of interest, and then growing the transfected host cells under conditions which allow for expression of the new recombinant protein. Certain bacterial host cell systems can be used to produce large quantities of recombinant proteins which are normally available in limited quantities from natural sources.
The procaryote Escherichia coli is a bacterium which has been studied extensively. E. coli. is commonly selected for use in high expression host cell systems, in part, because E. coli. cells tend to be more amenable to production of extremely large quantities of recombinant protein. Host cell systems employing eucaryotic host cells and yeast host cells generally fail to produce recombinant protein in the tremendous quantities generated in the high expression host cell systems like E. coli. Moreover, development of high cell density fermentation processes has resulted in increased volumetric productivity of recombinant products in E. coli. Yee and Blanch, Biotechnology and Bioengineering, 41: 221-230 (1993).
The fermentation processes used to produce recombinant proteins in host cell systems, like the E. coli. system, are carried out in finite physical containers (i.e. fermentors, reactors). Stirred tanks represent the most popular geometry of fermentors, although an increasing number of other physically shaped vessels are being developed. Modes of fermentor operation may fall into any of the following categories: (1) discontinuous operation (batch process), (2) continuous operation, or (3) various types of semi-continuous operations such as the fed-batch process.
Depending upon the mode of operation and host cell system being employed, a defined balanced batch and/or feed medium must be devised which will allow for cell growth and expression of the recombinant protein. The defined medium is termed “minimal” if it only contains the nutrients essential for growth. For the E. coli system, the minimal media must include a source of carbon, nitrogen, phosphorus, magnesium, and trace amounts of iron and calcium. Gunsalus and Stanter, The Bacteria, Vol. 1, Chapter 1 Academic Press Inc., N.Y. (1960). Most minimal media use glucose as a carbon source, ammonia as a,nitrogen source, and orthophosphate (e.g. PO4) as the phosphorus source. The ideal nutrient media for cell growth would include the exact amount of each nutrient that is consumed during cell growth, such that no nutrients accumulate to inhibitory levels, nor do the cells become starved of any nutrients. Thompson et al., Biotechnology and Bioengineering, 27: 818-824 (1985). A theoretically balanced minimal nutrient medium for E. coli has been devised previously for use in low cell density shake-flakes (cell densities up to 1.5 g cell dry weight/liter). Neidhardt et al., Journal of Bacteriology, 119: 736-747 (1974).
In addition to the chemical composition of the media, the effects of several other environmental parameters such as pH, time, cultivation temperature, and partial pressure of dissolved oxygen must be carefully considered. For example, the optimal pH for growth in E. coli is pH=7.0. During the fermentation process, pH of the media may be altered due to consumption of ammonia, or microorganism synthesis of certain metabolic products, e.g., acetic acid and lactic acid. Since altered pH may be unfavorable for optimal cell growth, it is critical to maintain the medium at a certain pH and this can be achieved by acid and base addition. The pH and other process parameters can be monitored manually or by automatic devices.
High cell density fermentations (i.e., those which achieve cell densities >20 g cell dry weight/liter) must employ a concentrated media. Operators performing high cell density fermentations have found that when working with concentrated nutrient medias, precipitates form when the solution containing the phosphate is mixed with the solution containing the other nutrient components. The precipitates that form in the nutrient media involve precipitation of orthophosphates and include NH4MgPO4, (Mg)3(PO4)2, and metallo-phosphates of the form (Me)n(PO4)m (where Me=Fe, Ca, Zn, Cu, Co). These compounds have very low solubilities in water. Dean, John A., Lange's Handbook of Chemistry, 12th edition, McGraw-Hill, New York, pages 7-12 (1979). The amount of precipitation can vary depending upon pH, glucose concentration, and concentration of the media components.
Precipitate formation can lead to a number of problems in feed medium and in the fermentor. For example, precipitates in the feed medium can lead to a non-homogeneous feed supply (due to settling of precipitate in feed vessels or supply lines), and starvation of the cells for critical nutrients that are no longer soluble. The precipitates can also abrade feed pumps and piping and possibly clog the feed lines altogether.
In the fermentor, precipitation will occur if the media is not perfectly balanced for cell growth. Precipitation in the fermentor can cause clogging of the air sparger in the fermentor, lead to nonhomogeneous mixing (i.e. precipitate settles in lower levels of the fermentor) and reduce the availability of soluble nutrients to the cells. The concentration of nutrients available to the cells becomes dependent on the rate of nutrient loss due to precipitate formation compared to the rate of nutrient uptake by the cells. These effects can be compounded by the automatic addition of acid and base for pH control. All of these conditions reduce the reproducibility of the fermentation process. Furthermore, the presence of precipitates can impact protein purification operations and force the use of extra purification process steps in order to separate the precipitate from the product.
In the commercial setting, in order for the fermentation process to be practical, the precipitation problem must be alleviated. One suggested way to prevent nutrient precipitation in minimal medium is the addition of EDTA and citrate in order to chelate metal ions in the nutrient media. Pirt, S. J., Principles of Microbe and Cell Cultivation, page 134 (1975). However, the need to add chelating agents is not desirable because the agents are not metabolized. Consequently, they accumulate and increase the osmolarity of the cellular environment. High osmolarity has a detrimental effect on cellular metabolism. Gouesbet et al., Journal of Bacteriology, 175: 214-221 (1993). High concentrations of chelating agents can also damage cell membranes. Ryan et al., Biotechnology and Bioengineering, 37: 430-444 (1991).
In German Patent Application No. 290,212 A5 (filed Jul. 28, 1988), Riesenberg et al. describe a procedure for the preparation of a glucose minimal medium for use in mass culture fermentation of E. coli for obtaining recombinant DNA products. The minimal media described by Riesenberg et al. was designed to overcome the heavy precipitation problems normally encountered when working with such medias. The media described in the application utilizes orthophosphates as the phosphorus source and does not completely eliminate nutrient precipitation; but rather, is said to exhibit only low turbulence due to light precipitate formation.
Aside from the discussion above, nothing can be drawn from the literature concerning preparation of medias for high cell density fermentations which effectively eliminate the nutrient precipitation problem. A need still exists for a method for reducing precipitation in a batch and/or feed media which contains no precipitation when all components are mixed (mixed at neutral pH for E. coli.), and which assures that no precipitate will form in the fermentor during processing. The present invention provides such a method by using a sodium phosphate glass as the source of phosphorus in the concentrated nutrient media. Unlike the methods suggested in the cited references above, the methods of the present invention provide the triple advantage of: (1) allowing for the design of a concentrated, completely balanced batch and/or feed media containing no precipitate; (2) being capable of being metabolized by E. coli; and (3) allowing for increased cell density and growth rates. The practical methods of the present invention are useful in a variety of bacterial fermentations, especially high cell density fermentation processes.